Neuroinflammatory responses may be dependent on the initiation of innate immune responses triggered by the stimulation of intrinsic brain cells by pathogen-associated molecular patterns (PAMPs), repeated structural motifs generated by microbes that are not normally found in the host or by debris from apoptotic or necrotic cells following injury. One group of receptors that recognize viral PAMPs is the toll-like receptor (TLR) 9 family, which is composed of TLR7, TLR8 and TLR9. These receptors form a closely related family that are highly homologous, confined to the endosomal membranes and recognize nucleic acids that are encountered in these compartments. TLR7 and TLR8 recognize single stranded RNA (ssRNA) from a number of virus families, while TLR9 recognizes unmethylated DNA containing CpG motifs (CpG-DNA) present in bacteria as well as DNA viruses. There are several recent studies demonstrating that TLR9 family members play an important role in neuropathogenesis, including our study demonstrating that TLR7 plays a role in retrovirus-induced innate immune responses in the CNS (Lewis, et.al. 2008, J. Neurovirology). Additionally, TLR7 and TLR9 agonists are being studied as potential therapeutics for multiple diseases in the brain, ranging from virus infections to the treatment of brain tumors. However, there is a lack of basic understanding of which cell types in the brain respond to stimulation of TLRs, as well as the pathways of neuroinflammation, neuroprotection and/or neuronal damage induced when these TLRs are activated. Understanding the similarities and differences of TLR7, TLR8 and TLR9-induced cell activation in the brain is important for understanding viral pathogenesis as well as potential use of TLR agonists in the treatment of neurological diseases. Our laboratory has focused on understanding the response of intrinsic brain cells following TLR activation and determining the downstream effects of TLR activation on neuroinflammation and neuropathogenesis. In Fy09, our studies have primarily focused on 1) determining the ability of resident brain cells including astrocytes, microglia and neurons to respond to TLR-specific agonists and determine the effect of stimulation on survival, function and innate immune responses 2) comparing the neuroinflammatory and neuropathogenic responses of the TLR7/9 agonists in vivo (3) analyzing the interaction between TLR7 and TLR9 agonists and/or receptors in regulating innate immune responses in the CNS. We have found that TLR7 and TLR9 agonists induce similar innate immune responses in glial cells in vitro, but differ vastly in their neuroinflammatory properties in vivo. Agonists of TLR9 were shown to induce a pronounced and robust neuroinflammatory response when administered to neonates by intracerebroventricular inoculation, and could induce morbidity and mortality in mice. These results contrast substantially to our previous studies with TLR7 agonists, which induce only mild inflammatory responses and no clinical response following an identical inoculation procedure (Butchi et. al. 2008, J. Immunology). Further studies are needed to determine the differences between TLR7 and TLR9 agonist- induced responses in order to better understand how activation of TLR9 in the CNS can induce neurological damage. We also identified interactions between agonists, with TLR7 agonists inhibiting TLR9 agonist-induced responses both in vitro and in vitro.